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March 10, 2021Advanced Algal SystemsLieve M. LaurensNational Renewable Energy Laboratory

BETO 2021 Peer Review1.3.5.270 Rewiring Algal Carbon Energetics for Renewables (RACER)

NREL | 2

Project Overview

The primary goal of the RACER consortium was to improve biofuel intermediate productivity of Desmodesmus armatus by addressing critical carbon conversion efficiencies towards a trifecta of fuel intermediates, ethanol or BDO, lipids and green crude oils

NREL | 3

Project Overview

Goals: Improve the overall carbon-to-fuel intermediate productivity for a biorefinery using D. armatus as a production species to reach at least 3,700 gal acre-1 yr-1

1. Improvements in photosynthetic carbon conversion efficiency through random mutagenesis and targeted engineering

2. Cultivation management advances through implementation of informed permutations of operations and nutrient management

3. Tailoring and optimizing conversion processes to extractable lipids, carbohydrate-derived fuel intermediate fermentation, and HTL biocrude from protein residue

Impact: Carbon conversion efficiency improvements by coordinating photosynthesis and carbon sink engineering is the basis of biomass accumulation and biofuels production, core to BETO’s AAS programOutcome: This project has studied a high-impact holistic process integration to demonstrate that improvements in strain engineering, cultivation operations and conversion engineering, can yield productivity improvements and cost reductions

Aim: Accelerate carbon to product pathwaysToday: No integrated path to targeting algae improvements with conversion in mindImportance: Holistic conversion relation to biomass composition sheds light on future algae challengesRisk: Relying on metabolic phenotype risks cascading effects

1. Photosynthesis mutant selection, carbon sink targeted engineering

2. Operations, e.g. pest management and harvesting conditions

3. Fermentation and pretreatment optimization, add mannose utilization to BDO strain, inhibit growth for yeast EtOH fermentation, optimize HTL extraction and nutrient recycling

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Value Proposition• Focus on strain development targeting

holistic carbon conversion efficiency (CCE) through processing

• Position for future emphasis on carbon assimilation to products and carbohydrates

Key Differentiators• Unique integration of upstream strain

engineering with downstream biomass conversion

• Connection to full project portfolio and SOT cultivation and conversion

NREL’s Bioenergy Program Is Enabling a Sustainable Energy Future by Responding

to Key Market Needs

Market Trends

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1. Strain Development2. Cultivation3. Conversion4. Technoeconomic &

Lifecycle Analysis

1. Management• Four subtasks aligned with project objectives, with monthly

project meetings and quarterly all-hands meetings• Progress tracked through modular process CCE

improvements, related to TEA/LCA• Commercialization strategy: Leverage and integrate with

BETO’s strategy for sustainable aviation fuels and chemicals and lipid and carbohydrate upgrading

• Deliverable: Yield and performance data on fuels and chemicals production available to community/industry

Lou Brown (SGI)Shaun Bailey (SGI)Rebecca White (Qualitas)Colin Beal (TEA consultant)Nancy Dowe (NREL Fermentation)Olaf Kruse (Bielefeld)Thomas Sharkey (Michigan State)

Advisory board:

Risk: Focused work on one species constrains integrated approach and risks associated with each conversion step requires flexible engineering customization approach

4. Computational TEA &LCARyan Davis, Jason Quinn

3. Pretreatment Engineering, Fermentation, ExtractionNick Nagle, Ken Reardon, Yat-Chen Chou, Tao Dong

2. Algae cultivation, data analysisJohn McGowen, Jessica Forrester

1. Genetic engineering, mutagenesis, physiologyEric Knoshaug, Damien Douchi, Graham Peers, Max Ware, Matt Posewitz, Melissa Cano

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2. ApproachObjective 1: Strain Development

1.1. Metabolic Engineering Tool Development1.2 Photosynthesis Engineering for Reduced Alternate Electron Transport1.3 Metabolic Rearrangement for Increasing Carbon Storage Sinks1.4 Engineering for Reduced Dark Respiration and Excreted Carbon Losses

Objective 2: Pond Operational Management2.1 Increased productivity through nutrient and pond operational management2.2 Submission of TERA Protocols

Objective 3: Conversion to Fuel Intermediates3.1 Pretreatment for Increased Feedstock Recovery3.2 Improved fermentation carbon conversion efficiency3.3 Optimize lipid extraction from fermentation stillage/slurry3.4 Optimization of HTL conversion of protein-rich residue

Objective 4: Techno-economic and Sustainability Analysis

✓

✓

✓✓✓✓

✓

✓--

-

Missed milestones due to genotype/phenotype instability Rescoping to incorporate new milestones for in-depth

comparative genetics

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2. Approach: Challenge Identification

Critical Success Factors Risk Strategy

Establish efficient metabolic engineering tools

Transient unstable expression of transgenes

Build robust strain development toolbox

Demonstrate improvements in overall productivity and fuel yields for D. armatus

Demonstrate physiological phenotype improvement

Variable gene expression may not lead to measurable improvement in cell carbon physiology

Engineering multiple targets using range of promotors and gene-expression elements and developing strong screening tools for mutagenesis approach as alternative for identifying improved strains

Pretreatment of biomass to supply conversion process with high yielding fractions

Biomass component interactions during acid pretreatment, biomass compositional variability

Optimization of small-to-large scale translation of pretreatment with different biomass material

Demonstrate carbon conversion improvements to biofuel streams

Liquors generated vary based on conversion conditions thus yielding different compositional fractions

Use optimized conversion or pretreatment approach at sufficiently large scale from one biomass harvest to supply consistent material to downstream conversion

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3. Impact• Increasing biofuel-intermediate yields by

removing key barriers that currently limit overall process carbon efficiency and biomass productivity

• Dissemination of results: 7 publications, 1 patent application, ranging from genetic engineering of non-model algae, nutrient recycling of products after HTL

• Transfer of technologies to other projects in algae projects portfolio:

– Genetic engineering applied to newly started project

– Fluazinam cultivation pest control to DISCOVR

– Conversion and BDO fermentation novel organisms to CPR

• Summer productivity of D. armatusincluded in the FY18 productivity SOT assessment

• Pretreatment and BDO fermentation yields included the conversion of sugars, including mannose, was included in the FY18, FY19 and FY20 SOT

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Objective 1: Strain Development4. Progress and Outcomes

Carbohydrates FAME

WT 32.6 ± 1.76 6.3 ± 0.08

1fc 38.44 ± 2.18 6.2 ± 0.19

2fc 36.7 ± 1.09 7.43 ± 0.18

8fc 38 ± 0.43 6.6 ± 0.45

7A6* 35.32 ± 1.48 6.75 ± 0.56

• Developed genetic engineering tools for D. armatus a unique and resilient non-model alga

• Selected and engineered mutants with distinct metabolic phenotype (increased carbohydrate and lipid content) and indoor growth rate improvements

• Discovered genetic machinery unique to D. armatus phenotype/genotype instability

[Knoshaug, E.P., 2019, Microbiol Resour Announc 9:e00896-19. https://doi.org/10.1128/ MRA.00896-19, Ware, M.A. et al. 2020, Plant Phys., 183, 1735-1748 | Ware, M. A., et al., 2020, Algal Research, 51, 102028; Douchi, D., et al., 2021, Algal Research, 53, 102152 | Mosey, M., et al., 2021, Algal Research, in press]

Spontaneous mutations appear in chloroplast genome after introduction of atrazine resistance marker mutation in PsbA

#11

#12

WT

plasmid

17% improvement in growth rate for 1FC + 18% increase in carbohydrates

*Calvin cycle overexpressor strain

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4. Progress and OutcomesObjective 2: Pond Operational Management

• Pioneered and mainstream implemented the use of Fluazinam pesticide (now routinely applied in DISCOVR consortium SOT trials)

• 27% increased productivity by increasing dilution rate, demonstrated in FY19, relative to FY18 baseline

• 10.9% improvement in productivity for 1FC mutant (September 2019 outdoor growth)

• 50 kg biomass produced for mutant and WT strains for conversion studies

27% improvement through dilution rates

11% improvement in outdoor productivity for 1FC mutant

[John McGowen, ASU]

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4. Progress and OutcomesObjective 3: Conversion to Fuels and Products

Engineered mannose utilization in Zymomonas BDO strain, 101% yield to 2,3-butanediol and acetoin based on glucose and mannose in hydrolysate (Ca(OH)2 neutralized)

• Pretreatment with enzymatic hydrolysis pioneered to over 95% glucose release

• Increased ethanol fermentation CCE by growth inhibition (comp P, comp Tp*)

• Showed 100% yield on BDO from algae hydrolysate after Zymomonas mannose utilization engineering

• > 92% lipid extraction efficiency (KeyLeaf) with 20% increased bio-oil yield from HTL (up to 38.8%)

• Recycled N from HTL aqueous phase**, showing a closed loop production-conversion-nutrient recovery study

[*Huang, Reardon, record of invention filed][** Chen and Laurens, 2019, Algal Research, 46, 101776]

DW (mg) EtOH yield (g/g)Control 3.6 ± 0.29 0.39 ± 0.01Comp P (0.2%) 2.57 ± 0.05 0.41 ± 0.01Comp Tp (0.2%) 1.83 ± 0.09 0.41 ± 0.01Continuous-flow prod 20% higher than free-cell batch fermentation of D. armatus hydrolyzate)

[Yat-Chen Chou, Min Zhang, NREL]

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4. Progress and OutcomesObjective 4: Technoeconomic and Sustainability Analysis

CAP Conversion Process Model

Inoculum System

Evaporation

Dilute Acid Hydrolysis

Solvent Extraction

HTL

Algae Growth CO2

(from fluegas carbon

capture)

Makeup nutrients

Recycle nutrients + CO2

ResidualBiomass

Lipids

Recycle water

Makeup water(groundwater pipeline)

CentrifugeMembranesSettlingSugar

Fermentation

Biocrude

Algal Biomass Production Process Model

Heat

Blowdown

Utilities and

Storage

Distillation

Hexane

Acid +Caustic

EthanolSteam Vent CO2

NGBlue – Inside RACER R&D scopeGrey – Outside RACER R&D scope

SLS

EtOH

BDOSolids

BDO(Aq.)

Aspen model complete with residue HTL integration

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4. Progress and Outcomes

22% reduction in MFSP demonstrated (intermediate verification), projected 40% if 17g/m2/day annual average productivity can be achieved

*Final fuel yield estimated > 3,700 gal/acre-yr (both with BDO and EtOH as fermentation products)

[Ryan Davis, NREL, Jason Quinn, Peter Chen, CSU]

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Impact• Carbon conversion efficiency improvements by

coordinating photosynthesis and carbon sink engineering for sustainable fuel production

• Improve basis of biomass accumulation and biofuels production, with TEA/LCA impact assessment

• Disseminate findings across collaborative portfolio

Progress & Outcomes• Discovery and documentation of genome instability

of non-model alga, D. armatus• Outdoor growth improvements >27% increased

productivity, with and 11% with mutant deployment• Improved carbon conversion to ethanol and 2,3

BDO as platform chemical and fuel intermediate• Projected > 20% decrease in MFSP after

integration of project deliverables

NREL’s Bioenergy Program Is Enabling a Sustainable Energy Future by Responding

to Key Market Needs

Summary

Management• Critical contribution to bioeconomy through

integrated objective product demonstration towards cost, value and feasibility demonstration

Approach• Coordinating photosynthesis engineering with

carbohydrate sink manipulation in non-model alga D. armatus

• Cultivation management for outdoor productivity improvements of selected cultivars

• Conversion optimization to biofuels and products

• TEA/LCA impact analysis for delivery and integration

Holistic process integration demonstrating improvementsin strain engineering, cultivation operations and conversionengineering, towards yield increase and cost reduction

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Timeline• 10/1/2017• 3-year competitive award• 95% complete

FY20 Active Project

DOE Funding

$292K $2.3M

Project CostShare

$315K

Project GoalImprove biofuel intermediate productivity of the commercially-relevant Desmodesmus armatus by addressing critical carbon conversion efficiencies towards a trifecta of fuel intermediates; ethanol or butane diol, lipids, HTL green crude oils

End of Project MilestoneAchieve overall biofuel intermediate yield of over 3900 gal acre-1 yr-1 based on both carbon assimilation and conversion improvements (FY20 MYP goal)

Project Partners*- Arizona State University (16%)- Colorado State University (26%)- KeyLeaf (2%), School of Mines (5%)- Sapphire Energy (cost share partner)

Funding MechanismFY17 ABY2 FOA, funded in FY18.

Quad Chart Overview

Barriers AddressedAft‐A. Biomass Availability and Cost, Aft‐C. Biomass Genetics and Development, Aft‐E. Algal Biomass Characterization, Quality, and Monitoring, Aft‐I. Algal Feedstock On‐Farm Preprocessing Aft‐J, Resource Recapture and Recycle

www.nrel.gov

Thank You

This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

Lieve.Laurens@nrel.govwww.nrel.gov/bioenergy/algal-biofuels.html

NRELEric KnoshaugDamien DouchiBo WangNick NagleTao DongYat-Chen ChouMin ZhangAmbarish NagStefanie Van WychenAndy PolitisSteven RowlandRyan HeroldMegan MoseyRyan DavisJenifer MarkhamPhilip Pienkos

CSUKen ReardonXingfeng HuangGraham PeersMax WareLaura HantzisJason QuinnPeter ChenJuan VenegasASUJohn McGowenJessica ForresterHenri GerkenCSMMatthew PosewitzMelissa CanoAmy AshfordKeyLeafRick GreenUdaya WanasundaraSapphireCraig BehnkeChris Yohn

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Additional Slides

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Publications, Patents, Presentations, Awards, and Commercialization

1. Chen, P. H., Venegas, J. L., Rowland, S. M., Quinn, J. C., Laurens, L. ML. (2020) Algae Nutrient Recycle from Hydrothermal Liquefaction Aqueous Phase through a Novel Selective Remediation Approach” Algal Research, 46, 101776

2. Knoshaug, E.P., Nag, A., Laurens, L. ML., 2019, Draft genome and chloroplast sequences of the biofuel-relevant microalga Desmodesmus armatus, Microbiology Resource Announcement, 9:e00896-19. https://doi.org/10.1128/ MRA.00896-19.

3. Maxwell A. Ware; Darcy Hunstiger; Michael B. Cantrell; Graham Peers, “A Chlorophyte alga utilizes alternative electron transport for primary photoprotection”, 2020, Plant Physiology, 183, 1735-1748

4. Ware, M. A., Kendrick, J. M., Hantzis, L. J., Peers, G. A fluorescence-based approach to screen for productive chemically mutagenized strains of Desmodesmus armatus, 2020, Algal Research, 51, 102028

5. Douchi, D., Mosey, M., Astling, D. P., Knoshaug, E. P., Nag, A., McGowen, J., Laurens, L. ML., Nuclear and chloroplast genome engineering of a productive non-model alga Desmodesmusarmatus: Insights into unusual and selective acquisition mechanisms for foreign DNA, 2021, Algal Research, 53, 102152

6. Mosey, M., Douchi, D., Knoshaug, E. P., Laurens, L. ML., Current Review of the Successes and Hurdles of Genetic Engineering of Non-model Algae, 2021, Algal Research, in press

7. Yeast quorum-sensing molecules reduce the cell growth of Saccharomyces cerevisiae and improve ethanol yield during fermentation (In preparation)

8. Provisional patent filed on quorum sensing molecules to improve fermentation CCE (CSU)

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Responses to Previous Reviewers’ CommentsWeakness: • They note in the presentation on slide 8 that they are going to increase productivity through nutrient and pond operational management, and later on slide 12 that they did a baseline outdoor cultivation in the summer of 2018. Were any lessons gleaned from this cultivation that could be used for the future cultivations with the genetically improved strains?

The goals of the outdoor cultivation baseline productivity experiments were to establish a seasonal average productivity for D. armatus at the AzCATI testbed location. This was expected to be different from the Las Cruces, NM testsite location, where the strain was grown for multiple consecutive years. In terms of lessons learned, the cultivation of D. armatus outdoors appeared to be robust in the first year of this project, but also prone to contamination with chytrids. This has promted us to implement a procedure for pond crash mitigation by pesticide application, which has yielded promising results. Until the improved strains become available, we are testing the impact of different cultivation conditions, e.g. semi-continuous cultivation at different densities, on the biomass productivity and composition.

Weakness: It was unclear what characteristics would be considered 'desireable' from a TEA standpoint. For example, mutagenesis work identified a mutant with increased lipid production, but it was unclear if the TEA would benefit from such a mutant.

While prior NREL TEA work has identified lipid content as a strong cost driver, particularly for fuel-focused conversion pathways included in the RACER project as a whole, in the context of this entire project, changes to biomass composition towards carbohydrate or lipid content increases are of interest to the overall biofuel intermediate yields. Work is ongoing both within the RACER project and under core BETO Platform efforts to specifically exercise NREL TEA models to identify cost versus value tradeoffs between biomass growth and composition, and the resultant implications on yields and fuel selling prices.

Weakness: Roll-out plan is focused on the genetic improvements that are being made to improve strains. What process improvements are being utilized to improve biomass productivities at scale? The methods for “increased productivity through nutrient and pond operational management” are not thoroughly clarified and should be seriously considered before large-scale implementation to ensure that ideal conditions are determined to produce optimum biomass, lipids, and carbohydrates in a production timely manner.

We believe that the biggest obstacles to outdoor cultivation improvements are related to creating stable mutant strains that show improvement in (mimicked) outdoor conditions. The ongoing experiments at the AzCATI testbed continue to collect outdoor data by comparing different cultivation conditions, in particular semi-continuous harvesting strategies (harvest frequency, biomass concentration and nutrient delivery permutations). Our primary target for the current outdoor experiments with the wild type strain is maximizing productivity, with continuous measurement of biomass composition. This will ultimately achieve a strong experimental basis to rapidly compare improved cultivars over the summer of FY19.

Weakness: • Growth parameters need to be optimized. • Strain has been reported to have 10% lipid during mid phase but increases to 20% after 13 days of induction. A large-scale production site will not be viable if induction to reach 20% lipids takes 2 weeks. Modes of induction to reach high lipids more rapidly should be considered. • Steps are delineated for how strains are going to be genetically modified for improvements, but no steps are noted for how strains are going to be enhanced through “nutrient and pond operational management” (slide 8). What approaches are being utilized to boost productivity for these strains from an operational standpoint?

We appreciate the comments relating to the cultivation work but wanted to clarify that we are not planning to include a dedicated lipid induction phase during the outdoor cultivation experiments. The targeted metabolic engineering tools that are in the process of being developed focus on targeting central carbon assimilation and carbohydrate storage, so in our targeted effort, it is much more likely that carbohydrates will be accumulated, and lipids are projected to either stay constant or increase slightly as an overflow storage for metabolic energy. The experimental outdoor cultivation approach on nutrient and pond operational management will include different permutations on the harvesting strategy, e.g. keeping the cultures at high cell density but low depth will increase light stress and thus has the potential to rapidly shift the composition, while also maximize biomass productivity, both of which would be highly beneficial in the overall integrated process operations.

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Approach

• Go/No-go Decision point (FY19)

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Key TechFin Cultivation parameters in put to TEA

22

TechFin: Key TEA model outputsParameter: EtOH [BDO]

Baseline Baseline Update

Intermediate Intermed. Update

Final

Biomass yield (tonne/yr AFDW) 87,338 87,338 102,361 102,361 131,608

Biomass selling price ($/ton AFDW) $935/ton $909/ton $810/ton $783/ton $651/ton

Intermediate fuel yield (GGE/ton AFDW) 102 76 [80] 109 88 [87] 120

Lipid output (MM gal/yr) 4.4 1.8 5.1 2.2 6.1

Ethanol/BDO output (MM gal/yr) 3.3 3.0 [2.9] 4.8 3.2 [2.8] 8.0

HTL bio-crude output (MM gal/yr) 3.7 4.0 4.4 6.4 6.7

Overall intermediate fuel yield (gal/acre-yr)

2200 1727 [1719] 2771 2288 [2197]

4033

Integrated conversion CAPEX (TCI, $MM) $121 $144 [$151] $132 $178 [$185]

$155

MFSP ($/GGE intermediates) $11.63 $15.31 [$14.97]

$9.67 $12.00 [$12.39]

$7.07

• Final MFSP projected at $7.07/GGE (~40% improvement over original baseline or ~50% over updated baseline on EtOH) – will not achieve “economic viability” by end of project, but this was never expected (not until beyond 2022) – that would require >25 g/m2/day and inclusion of coproducts (at expense of fuel yields)

• Final target case achieves primary FOA objective: fuel yield target >3700 gal/acre-yr• Updated intermediate case achieves Phase I TEA objective: >10% MFSP improvements over updated (re-

cast) baseline (both EtOH and BDO)

Lower net fuel yield: lower lipid content, higher

carbs offset by SLS losses + lower fermyields; higher HTL

yields

Higher MFSP due to lower overall

yields, higher processing costs

from model refinements

Higher lipid extraction yield,

higher carbs offset by lower

prt yields, higher HTL yield

22% and 17% lower MFSP for

EtOH and BDO vs updated baseline

– lower MBSP, higher yields

23

Metric Baseline Baseline Update Intermediate Intermed.

Update Final

Pretreatment

Solids loading (wt%) 20% 20% 20% 18% 20%

Temperature (°C) 150 150 150 170 150

Acid loading (wt% vs feed liquor) 2% 2% 2% 3% 1%

Fermentable sugar release (wt% of initial carbs) 74% 74% 80% 56% glucose,

39% mannose 90%

Fermentation

Monomeric sugar utilization; EtOH [BDO] 95% 95% [26%] 95% 95% [98%] 98%

Metabolic yield (g/g utilized sugars, % theor); EtOH [BDO] 86% 80% [73%] 86% 88% [90%] 92%

Batch time (days); EtOH [BDO] 1.5 1.5 [3.5] 1.5 1.5 [2.2] 1.5

Lipid Extraction

Solvent loading (solvent/dry biomass ratio, g/g) 5.9 5.9 5.9 3.0 5.0

Total FAME lipid extraction yield 87% 87% 90% 92% 95%

HTL Conversion of Residue

Biocrude yield (wt% of organic feed) 23.3% 23.8% 26.2% 31.2% 35.7%

N/P recycle to cultivation (% of HTL feed) 63% / 50% 63% / 50% 63% / 50% 63% / 50% 63% / 50%

TechFin: Conversion: Key modeling inputs• Baseline: Prior NREL SOT

data (HTL assumed)• Baseline update: Corrected

EtOH yield based on CSU S. cerevisiae free cell ferm. + NREL HTL baseline data

• Intermediate/Final: Original verification goals

• Intermed. update: RACER Phase 1 demonstrated

NREL CAP Design Report:

http://www.nrel.gov/docs/fy14osti/62368.pdf

CSU free cell fermentation

High recalcitrance with D. armatus

-EtOH: immob. cells-BDO: Zymoimprovements, lower toxicity

POS extr. yields w/ hexane

New Keyleaf extraction data

NREL | 24

Overexpression targets

#1 = Levansucrase in the vacuole – no transformation yet#2 = Cellulose synthase – no transformation yet#3 = Fbp-Sbp (D-fructose 1,6-bisphosphatase class 2/sedoheptulose 1,7-bisphosphatase)#4 = Levansucrase in the cytoplasm – 2 transformant lines#5 = PGM (Phosphoglucomutase) – 2 transformant lines#6 = g7207 - 7 transformant lines#7 = fbaA (Fructose-bisphosphate aldolase class 2)#8 = FSBP (Fructose-1,6-/sedoheptulose-1,7-bisphosphatase) – 15 transformant lines#9 = ADP-glucose pyrophosphorylase – no transformation yet#10 = Isoamylase – no transformation yet

*Calvin cycle carbon assimilation*Storage carbon sink

(WT control)

FSBP

cNAT (nourseothricin acetyl transferase) resistance marker confirmed insertion:

Growth rate comparison:

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Multivariate Analysis – ‘Omics’ Integration

Calvin cycle

CO2shuttle

Established RNASeq analysis pipeline for D. armatus for transcriptomics of Carbon Fixation pathway under abiotic stress

Lee, P., Astling, D., et al. (2018) in preparation

NREL | 26

Night-time Respiration – Starch Degradation

Starch degradation pathway model in Chlamydomonasand higher plants indicating starch phosphorylation at the core of regulating degradation flux

CO2 uptake/release in the headspace for three weeks of Scenedesmus acutus cultivation in programmable SAGE reactor indicating night-time respiration

Remains to be demonstrated whether D. armatus exhibits this starch metabolism

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Glucan Water DikinaseGenome mining of Desmodesmus armatus indicates the presence and similar structure of the gene for Glucan Water Dikinase (GWD) – elimination causes starch excess phenotype without impacting growth

28

Multivariate Analysis – ‘Omics’ Integration

Endoglucanase expression

Transcriptomics of Starch and Sucrose Metabolism in D. armatus

Established RNASeq analysis pipeline:

Starch synthesis

Glycogen synthesis

Glycan hydrolases

Lee, Astling, Laurens (2017) in preparation

29

Extracellular Gene Expression

Cation transporter

Endochitinase

Catalase peroxidase

Endoglucanase

Disulfide isomerase

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Engineering Z. mobilis for 2,3-BDO production from Algal Sugars

• Introduce mannose utilization pathway in Z. mobilis strain– Mannose utilization pathway will be introduced into Z. mobilis ZM4 (it

has previously introduced a different host)• Introduce 2,3 BDO producing pathway to mannose- utilizing Z. mobilis

strain – 2,3 BDO producing pathway has been engineered into glucose/xylose-

utilizing Z. mobilis strain. • Eliminate ethanol formation in the above 2,3 BDO producing/mannose-

utilizing Z. mobilis strain• Fermentation testing for 2,3 BDO production from algal biomass sugar

streams – comparing yields (g BDO / g sugar) against baseline of pure sugar

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Metabolic Engineering Zymomonas mobilis for Producing 2,3-BDO2,3-BDO: versatile chemical building block for producing solvents, chemicals, jet fuels and fuel additives

XyloseArabinose

Glucose

Ethanol

Entner-Doudoroff(ED) pathway

“Catabolic Highway”PPP

pathwayG3P

Pyruvate

2,3-Butanediol

Biomass after DMR pretreatment and enzymatic hydrolysis containing mixed C6/C5 sugars

BDO is less toxic

Anaerobic process for ethanolBut need to purge low level of air for oxidizing the excess NADH for BDO

Potential high TYR Upgrade to fuels and chemicals

Bdh butanediol dehydrogenase

Aldc acetolactate decarboxylase

Als acetolactate synthase

Glucose

XyloseArabinose

Ethanol 2,3-Butanediol

AcetaldehydeAcetolactate

R-Acetoin

Weisser, Peter, Reinhard Kramer and Georg A. Sprenger. Expression of the Escherichia coli pmigene, encoding phosphomannose-isomerase in Zymomonasmobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker. Appl. Environ. Microb. 62, 4155-4161. 1996

Addition of phosphomannoseisomerase (pmi) conveys mannose utilization

Mannose

Mannose

Mannose-6-P

Glucose-6-PFructose-6-P